Phosphoinositide 3-kinases are critical regulators of proliferation, motility, apoptosis and vesicular trafficking, and are constitutively activated in human cancers. Class IA enzymes are composed of a regulatory subunit (p85) that inhibits a distinct catalytic subunit (p110). The minimal fragment of p85 required for regulation of p110 is a coiled-coil region, the iSH2 domain, linked to a single SH2 domain (the nSH2 domain). Isolated iSH2 domains are sufficient to bind to p110, but inhibition additionally requires the nSH2 domain linked to the iSH2 domain. How does the nSH2 domain exert its effects? Using site-directed cysteine mutagenesis and EPR spectroscopy, the iSH2 domain has been demonstrated to be a conformationally rigid coiled-coil consisting of two 100 A-long antiparallel helices. This has led to a new model, in which the iSH2 domain binds p110, facilitating additional inhibitory contacts between the nSH2 domain and p110. This model is supported by recent structural studies defining a region of the distal iSH2 domain that makes a close contact with the nSH2 domain. Truncations of the iSH2 domain that remove this region abolish p85 inhibition of p110. The present proposal tests the hypothesis that the orientation of the nSH2 domain is a critical determinant of p110 activity, and that this orientation is maintained by specific contacts between the nSH2 domain and the iSH2 domain. Aim 1 will use NMR spectroscopy to define the structure of the nSH2-iSH2-cSH2 fragment of p85 in its basal state, when activated by phosphopeptides or oncogenic mutations, and when inhibited by serine autophosphorylation. Aim 2 uses EPR spectroscopy to examine the relative orientation of the nSH2 and iSH2 domains within the context of p85/p110 dimers, and to test the hypothesis that different activating inputs to p85 (binding of activated Cdc42 to the BCR domain versus binding of phosphopeptides to the SH2 domains) exert their effects through similar mechanisms. Aim 3 will use mass spectrometry-based oxidative foot printing to identify p85/p110 contacts, determine how these contacts are modulated by activators/inhibitors of p85/p110, and explore the tertiary organization of p85. Completion of these aims will greatly increase our understanding of the p85/p110 PI 3-kinase, and lead to mechanistic insights into its activation in human cancer.